With the advance in molecular medicine, our understanding of molecular biology of cancer initiation, progression and treatment have improved, resulting in better therapeutic approaches. However, since a neoplastic cell can be characterised as a de-differentiated normal cell with chromosome instability, novel mutations occur, making established anti-cancer drugs obsolete. Certain malignancies, including malignant mesotheliomas (MMs), cannot be treated at present (Robinson B W, Musk A W, Lake A (2005) Lancet 366, 397-408). Others, such as the HER2-positive breast carcinomas (Piccart-Gebhart M Jet al (2005) N Engl J Med 353, 1659-1672) rely on the very expensive Herceptine™ that shows considerable cardiotoxicity. Thus, new drugs and strategies that would overcome complications of current therapeutic approaches are required.
A novel concept has emerged whereby targeting of mitochondria has become increasingly recognised as a promising and highly effective anti-cancer approach (Don A S, Hogg P J (2004) Trends Mol Med 10, 372-378; Armstrong J S (2007) Br J Pharmacol 151, 1154-1165). The present inventors have recently proposed the term, “mitocans”, referring to small molecules with anti-cancer activity that induce apoptosis by destabilizing mitochondria in cancer cells (Neuzil J, Wang X F, Dong L F, Low P, Ralph S J (2006) FEBS Lett 580, 5125-5129; Neuzil J et al (2007) J Bioenerg Biomembr 39, 65-72; Neuzil J et al (2007) Mol Pharmacol 71, 1185-1199). The present inventors have grouped these molecules according to their mode of action, as seen in Table I (Neuzil Jet at (2007) Mol Pharmacol 71, 1185-1199).
TABLE IClassification of mitocans.ClassTypeExamplesIHexokinase3-Bromopyruvate (Ko Y H et al (2004)inhibitorsBiochem Biophys Res Commun 324, 269-275;Xu R H et al (2005) Cancer Res 65, 613-621)2-Deoxyglucose (Ko Y H et al (2004)Biochem Biophys Res Commun 324, 269-275)IIBcl-2/Bcl-xL Gossypol (Kitada S et al (2008) Blood (inBH3press))mimeticsAntimycin A (Tzung S P et al (2001) Nat CellBiol 3, 183-191)BH3I-2′ (Degterev A et al (2001) Nat CellBiol 3, 173-182)α-Tocopheryl succinate (Shiau C W et al(2006) J Biol Chem 281, 11819-11825)IIIThiol redoxIsothiocyanates (PITC) (Xu K, Thornalley P Jinhibitors(2001) Biochem Pharmacol 61, 165-177)Arsenic trioxide (Miller W H (2002)Oncologist 7, S14-19)IVVDAC/ANTLonidamine (Belzacq A S et al (2001)targeting Oncogene 20, 7579-7587)drugsArsenites (Don A S et al (2003) Cancer Cell 3,497-509)VElectron 4-OH retinamide (Hail N, Lotan R (2001) JtransportBiol Chem 276, 45614-45621)chain Tamoxifen (Moreira P I et al (2006) J BioltargetingChem 281, 10143-10152)drugsAntimycin A (Wolvetang E J, Johnson K L,Krauer K, Ralph S J, Linnane A W (1994)FEBS Lett 339, 40-44)α-Tocophelyl succinate (Dong L F et al(2008) Oncogene (in press))VILipophilic Rhodamine-123 (Lampidis T J, Bernal S D,cationsSummerhayes I C, Chen L B (1983) Cancer Restargeting 43, 716-720)innerF16 (Fantin V R et al (2002) Cancer Cell 2,membrane29-42)(KLAKKLAK)2 peptide (Ellerby H M et al(1999) Nat Med 5, 1032-1038)VIIDrugs Resveratrol (ATPase ?) (Zheng J, RamireztargetingV D (1999) Biochem Biophys Res Communother sites261, 499-503)Betulinic acid (Fulda S et al (1998) J BiolChem 273, 33942-33948), DCA (Bonnet S etal (2007) Cancer Cell 11, 37-51)
The list of mitocans currently includes 7 groups, each of them comprising agents with distinct activities, whereby causing mitochondrial destabilisation and the ensuing induction of the intrinsic apoptotic pathway (Neuzil J et al (2007) Mol Pharmacol 71, 1185-1199).
Mitocans are proving to be attractive for the treatment of cancer since some of these compounds are potent and selective anti-cancer agents with little effect on normal cells (Neuzil J et al (2007) Mol Pharmacol 71, 1185-119; Ko Y H et al (2004) Biochem Biophys Res Commun 324, 269-275; Bonnet S et al (2007) Cancer Cell 11, 37-51). Prime examples of such drugs include α-tocopheryl succinate (α-TOS), inducing selective apoptosis of cancer cells (Neuzil J, Weber T, Gellert N, Weber C (2001) Br J Cancer 84, 87-89; Neuzil J et al (2004) Curr Cancer Drug Targets 4, 267-284) as well as 3-bromopyruvate (3BP) (Ko Y H et al (2004) Biochem Biophys Res Commun 324, 269-275; Geschwind J F et al (2002) Cancer Res 62, 3909-3913), dichloroacetate (DCA) (Bonnet S et al (2007) Cancer Cell 11, 37-51) and β-phenylethyl isothiocyanate (PITC) (Trachootham D et al (2006) Cancer Cell 10, 241-252).
3BP inhibits hexokinase, an enzyme of the glycolytic pathway predominantly bound to the external face of mitochondria, and also inhibits the mitochondrial enzyme succinate dehydrogenase (SDH), suppressing cellular ATP production and mitochondrial respiration (Ko Y H et al (2004) Biochem Biophys Res Commun 324, 269-275; Xu R H et al (2005) Cancer Res 65, 613-621).
DCA appears to selectively target cancer cells by inhibiting the mitochondrial pyruvate dehydrogenase kinase (Bonnet S et al (2007) Cancer Cell 11, 37-51). PITC selectively kills cancer cells by causing mitochondrial generation of reactive oxygen species (ROS) (Trachootham D et al (2006) Cancer Cell 10, 241-252). All these examples of mitocans reflect an emerging group of compounds with substantial promise and a new direction for developing improved and highly selective novel anti-cancer drugs.
One group of mitocans includes pro-oxidant analogues of vitamin E (Wang X F, Dong L F, Zhao Y, Tomasetti M, Wu K, Neuzil J (2006) Vitamin E analogues as anti-cancer agents: Lessons from studies with α-tocopheryl succinate. Mol Nutr Food Res 50:675-685). The great promise of pro-oxidant vitamin E analogues, epitomized by α-TOS, as anti-cancer drugs stems from studies with experimentally contrived cancers, such as human xenografts growing in nude mice, where they have been shown to suppress malignancy (reviewed in Neuzil J, Tomasetti M, Mellick A S, Alleva R, Salvatore B A, Birringer M, Fariss M W (2004) Vitamin E analogues: A new class of inducers of apoptosis with selective anti-cancer effects. Curr Cancer Drug Targets 4:355-372). Such studies include colorectal (Neuzil J, Weber T, Gellert N, Weber C (2001) Selective cancer cell killing by α-tocopheryl succinate. Br J Cancer 84:87-89; Neuzil J, Weber T, Schroder A, Lu M, Ostermann G, Gellert N, Mayne G C, Olejnicka B, Negre-Salvayre A, Sticha M, Coffey R J, Weber C (2001) Induction of apoptosis in cancer cells by α-tocopheryl succinate: Molecular pathways and structural requirements. FASEB J 15:403-415; Weber T, Lu M, Andera L, Lahm H, Gellert N, Fariss M W, Korinek V, Sattler W, Ucker D S, Temman A, Schroder A, Erl W, Brunk U T, Coffey R J, Weber C, Neuzil J (2002) Vitamin E succinate is a potent novel anti-neoplastic agent with high tumor selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L) in vivo. Clin Cancer Res 8:863-869) and lung carcinomas (Quin J, Engle D, Litwiller A, Peralta E, Grasch A, Boley T, Hazelrigg S (2005) Vitamin E succinate decreases lung cancer tumor growth in mice. J Surg Res 127:139-143), melanomas (Malafa M P, Fokum F D, Mowlavi A, Abusief M, King M (2002) Vitamin E inhibits melanoma growth in mice. Surgery 131:85-91), as well as mesotheliomas (Tomasetti M, Gellert N, Procopio A, Neuzil J (2004) A vitamin E analogue suppresses malignant mesothelioma in a pre-clinical model: A prototype of a future drug against a fatal neoplastic disease? Int J Cancer 109:641-642; Stapelberg M, Gellert N, Swettenham E, Tomasetti M, Witting P K, Procopio A, Neuzil J (2005) α-Tocopheryl succinate inhibits malignant mesothelioma by disruption of the FGF autocrine signaling loop: Mechanism and the role of oxidative stress. J Biol Chem 280:25369-25376). α-TOS has also been shown to promote breast cancer dormancy (Malafa et al, 2000) and suppress colon cancer metastases into the liver (Barnett K T, Fokum F D, Malafa M P (2002) Vitamin E succinate inhibits colon cancer liver metastases. J Surg Res 106:292-298).
Although vitamin E (α-tocopherol, α-TOH) acts as a potent anti-oxidant in cells, α-TOS, an esterified, redox-silent and pro-oxidant analogue of vitamin E, has distinctive properties. In contrast to α-TOH, α-TOS acts as a strong cell stressor, causing rapid production of ROS in a range of different cancer cell lines (Neuzil J, Tomasetti M, Mellick A S, Alleva R, Salvatore B A, Birringer M, Fariss M W (2004) Vitamin E analogues: A new class of inducers of apoptosis with selective anti-cancer effects. Curr Cancer Drug Targets 4:355-372; Weber T, Dalen H, Andera L, Negre-Salvayre A, Auge N, Sticha M, Lloret A, Terman A, Witting P K, Higuchi M, Plasilova M, Zivny J, Gellert N, Weber C, Neuzil J (2003) Mitochondria play a central role in apoptosis induced by α-tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signaling. Biochemistry 42:4277-4291; Wang X F, Witting P K, Salvatore B A, Neuzil J (2005) α-Tocopheryl succinate induces apoptosis in HER2/erbB2-overexpressing breast cancer cells by signalling via the mitochondrial pathway. Biochem Biophys Res Commun 326:282-289; Swettenham E, Witting P K, Salvatore B A, Neuzil J (2005) α-Tocopheryl succinate selectively induces apoptosis in neuroblastoma cells: Potential therapy of malignancies of the nervous system? J Neurochem 94:1448-1456; Stapelberg M, Gellert N, Swettenham E, Tomasetti M, Witting P K, Procopio A, Neuzil J (2005) α-Tocopheryl succinate inhibits malignant mesothelioma by disruption of the FGF autocrine signaling loop: Mechanism and the role of oxidative stress. J Biol Chem 280:25369-25376). α-TOS also has the ability to bind to and inhibit Bcl-2/Bcl-xL (Dong L F, Wang X F, Zhao Y, Tomasetti M, Wu K, Neuzil J (2006) Vitamin E analogues as anti-cancer agents: the role of modulation of apoptosis signalling pathways. Cancer Therapy 4:35-46). Evidence to date suggests that the cancer cell-specific nature of α-TOS and the lack of toxic effect on normal cells occurs because normal cells are endowed with greater anti-oxidant defenses (Allen R G, Balin A K (2003) Effects of oxygen on the antioxidant responses of normal and transformed cells. Exp Cell Res 289:307-316; Safford S E, Oberley T D, Urano M, St Clair D K (1994) Suppression of fibrosarcoma metastasis by elevated expression of manganese superoxide dismutase. Cancer Res 54:4261-4265; Church S L, Grant J W, Ridnour L A, Oberley L W, Swanson P E, Meltzer P S, Trent J M (1993) Increased manganese superoxide dismutase expression suppresses the malignant phenotype of human melanoma cells. Proc Natl Acad Sci USA 90:3113-3117) and/or contain high levels of esterases that inactivate α-TOS by releasing the succinate moiety, thereby producing the redox-active, non-apoptogenic α-TOH (Fariss M W, Nicholls-Grzemski F A, Tirmenstein M A, Zhang J G (2001) Enhanced antioxidant and cytoprotective abilities of vitamin E succinate is associated with a rapid uptake advantage in rat hepatocytes and mitochondria. Free Radic Biol Med 31:530-541; Neuzil J, Tomasetti M, Mellick A S, Alleva R, Salvatore B A, Birringer M, Fariss M W (2004) Vitamin E analogues: A new class of inducers of apoptosis with selective anti-cancer effects. Curr Cancer Drug Targets 4:355-372; Neuzil J, Massa H (2005) Hepatic processing determines dual activity of vitamin E succinate. Biochem Biophys Res Commun 327:1024-1027).
Naturally occurring vitamin E consists of a mixture of eight compounds which differ by the methylation patterns of the chromanol ring (α-, β-, γ-, δ-tocopherol) and the number of double bonds of the phytyl side-chain (α-, β-, γ, δ-tocotrienol). The role of these molecules as lipophilic anti-oxidants in vitro and in vivo is widely accepted. In addition, the non-anti-oxidant properties of members of the VE family have also been investigated (Azzi A, Ricciarelli R and Zingg J M (2002) Non-antioxidant molecular functions of α-tocopherol (vitamin E). FEBS Lett 519:8-10).
The vitamin E molecule can be divided into three different domains. The Functional Domain (I) arises from the substitution pattern at position C6 of the chromanol ring. This position determines whether the molecule behaves as redoxactive or redox-silent, since a free hydroxyl group is essential for vitamin E to function as an anti-oxidant. The well documented anti-oxidant properties of the four tocopherol isomers resulted in their application in cancer clinical trials. None of these studies showed a positive outcome concerning the use of free tocopherols in cancer prevention (Pham DQ and Plakogiannis R (2005) Vitamin E supplementation in cardiovascular disease and cancer prevention: Part 1. Ann Pharmacother 39:1870-8). However, certain chemical modifications at C6 led to ethers (RO—), esters (RCOO—) and amides (RCONH—) that proved to be potent anti-neoplastic agents. See Table II below.
TABLE IIAnti-proliferative activity of vitamin E analogues.Compounds are sorted by the Signaling Domain.Ref (seeFunctionalSignallingHydrophobicIC50Cellthe listNrDomain I (R1)Domain IIDomain III (R2)[μM]typeof refs) 1  2  3  4  5  6  7  8  9 10 11   12 13 14 15 16 17 18     19   20           21   22   23         24 25 26 27   28 29     30 31−O2CCH2CH2COO— CH3COO— −O2CCH═CHCOO— −O2CCH2CH(CH3)COO— −O2CCH2(CH2)2COO— −O2CCH2CH(CH3)CH2COO— −O2CCH2C(CH3)2CH2COO— −O2CCH(CH3)2CH2CH2COO— H3COOCCH2CH2COO— −O2CCOO— −O2CCH2COO   −O2CCH2CH2CONH— −O2CCH═CHCONH— H3COOCCH2CH2CONH— +NH3—CH2COO— +NH3Lys(NH3)COO— Lys-Lys(Lys)COO— CH3O—     CH3CH2COO—   −O2CCH2CH2CH2O—           −O2CCH2O—   −O2CCH2—   (PEG)O2CCH2CH2COO—         −O2C(CH2)5COO— C2H5OOCCH2CH2COO— nicotinic acid −O2CCH2CH(SePh)COO—   all-trans retinoic acid 9-cis retinoic acid     HOPO2O— Toc-OPO2O—  43 a   22 b b b b b b c       13   2 >100 a   12 a a     d   e           f   15-20g   h         a a a ?   0,1-1 b     b bJurkat, HBT11, MCF7, MCF7-C3           B16-F1/ nude mice Jurkat, U937, Meso-2 MCF7     Jurkat     A549   LNCa P, PC-3 MDA-MB-453       MDA-MB-435, MCF7 MCF7   lung carcinoma cells/ nude mice C127I     prostate   NB4, HT93     RAS MC, THP-1Birringer et al, 2003             Kogure et a., 2005 Tomoc- Vatic et al, 2005 Arya et al, 1995   Neuzil et al, 2001b Yano et al, 2005 Wu et al, 2004; Nishika wa et al, 2003 Shun et al, 2004 Shiau et al, 2006 Youk et al, 2005       Kogure et al, 2004 Vraka et al, 2006 Makishima et al, 1996, 1998 Munteanu et al, 2004 32−O2CCH2CH2COO—50% of α-TOSJurkat, HBT1, MCF7, MCF7-C3, U937, Meso-2Birringer et al, 2003; Tomic- Vatic et al, 2005 33         34−O2CCH2CH2COO—         −O2CCH2CH(SePh)COO—a         bJurkat, HBT1, MCF7, MCF7-C3   prostateBirringer et al, 2003; Vraka et al, 2006 Vraka et al, 2006 35           36 37 38 39     40−O2CCH2CH2COO—           −O2CCH═CHCOO— −O2CCH2CH2CONH— −O2CCH═CHCONH— H3COOCCH2CH2COO—     HO—  66             49   20   9 a     bJurkat, HBT1, MCF7, MCF7-C3     Jurkat, U937, Meso-2       PC-3Birringer et al, 2003; Tomic- Vatic et al, 2005 Tomic- Vatic et al, 2005 Birringer et al, 2003 Galli et al, 2004aNo effect;binhibition of cell proliferation;cmuch more cytotoxic than α-TOS;dless effective than 54;ether analogue is less effective than α-TOS itself;f(comparable to α-TOS;gEC50[μg/ml];hmore efficient than α-TOS.
TABLE IIIAnti-proliferative activity of vitamin E analogues with a modifiedHydrophobic Domain.FunctionalSignallingHydrophobicCellNr.Domain I (R1)Domain IIDomain III (R2)IC50 [μM]typeRef41       42 43 44−O2CCH2CH2COO—       HO— −O2CCH2CH2COO— −O2CCH2CH2O—COO−       a       a 4-9 4-8Jurkat, HBT11, MCF7, MCF7-C3 LNCaP, PC-3Birringer et al, 2003   Shiau et al, 2006 45−O2CCH2CH2COO— 8-19 46CH3>10047+NH3Lys(NH3)COO—CH2—OH 194MCF7Arya e tal, 199548CH2—O-nC5H11  2249CH2—OC(O)nC4H9  1550CH2—O-cholic acid  451HO—CH2CH2COO−bPC-3Galli et al, 2004 52HO—CH2CH2COO−caNo effect;bweak inhibition at 50 μM;c82% inhibition at 10 μM.
TABLE IVAnti-proliferative activity of vitamin E analogues. Compounds aresorted by the Signaling Domain.FunctionalSignallingHydrophobicIC50CellNr.Domain I (R1)Domain IIDomain III (R2)[μM]typeRef.53         54   55HO—         CH3CH2COO—   HO—         210  14   110   a   bMDA-MB-435 MCF7   B16(F10)   A549   Jurkat, HBT11, MCF7, MCF7-C3Guthrie et al, 1997 He et al, 1997 Yano et al, 2005 Birringer et al, 2003 56HO— 4          15c   d        20neoplast Ic + SA mammary epithelial cells MCF7   Jurkat, HBT11, MCF7, MCF7-C3 B16(F10)Shah and Sylvester, 2005   He et al, 1997 Birringer et al, 2003   He et al, 1997 57−O2CCH2CH2COO—eJurkat, HBT11, MCF7, MCF7-C3Birringer et al, 2003fprostateVraka et al, 200658−O2CCH2CH(SePh)COO—fprostateVraka et al, 2006 59HO— 10   b    15cB16(F10)   MDA-MB-435, MCF7 MCF7He et al, 1997 Shun et al, 2004 Nesaretnam et al, 1998 60HO— 0.9B16(F10)He et al, 1997aCytotoxic in 0-40 μM range;bvery potent;ccomplete inhibition;dcomparable to α-TOS;e2-fold more potent than γ-tocotrienol;finhibition of cell proliferation.
The second domain, termed the Signaling Domain (II), exhibits some activities that are independent of the anti-oxidant properties of the tocopherols. These properties derive from the methylation pattern of the aromatic ring. For example, α-tocopherol has been reported to inhibit protein kinase C (PKC) by decreasing diacylglycerol (DAG) levels, while other tocopherols with similar anti-oxidant capabilities (e.g., β-tocopherol) do not inhibit PKC. Thus, the PKC inhibitory activity of α-tocopherol is independent of its anti-oxidant capacity (Tasinato A, Boscoboinik D, Bartoli G M, Maroni P and Azzi A (1995) d-α-Tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties. Proc Natl Acad Sci USA 92:12190-12194; Kunisaki M, Bursell S E, Clermont A C, Ishii H, Ballas L M, Jirousek M R, Umeda F, Nawata H and King G L (1995) Vitamin E prevents diabetes-induced abnormal retinal blood flow via the diacylglycerol-protein kinase C pathway. Am J Physiol 269:E239-246). In some cases, however, the biological activity of the various tocopherols is influenced by structural differences in the Signaling Domain, which do indeed have a profound impact on their anti-oxidant activity against certain species. γ-Tocopherol, for example, is a much better scavenger of reactive nitrogen oxide species (e.g., peroxynitrite) than α-tocopherol. Hence, the γ-molecule, which lacks a methyl group at C5, is readily nitrated at that site (Morton L W, Ward N C, Croft K D, Puddey I B. (2002) Evidence for the nitration of gamma-tocopherol in vivo: 5-nitro-gamma-tocopherol is elevated in the plasma of subjects with coronary heart disease. Biochem J. June 15; 364(Pt 3):625-8; Christen S, Woodall A A, Shigenaga M K, Southwell-Keely P T, Duncan M W, Ames B N. (1997) gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications. Proc Natl Acad Sci USA. April 1; 94(7):3217-22).
The lipophilic side chain of vitamin E isomers distinguishes between tocopherols with saturated isoprenyl units and tocotrienols with unsaturated isoprenyl units. The Hydrophobic Domain (III) determines whether the molecule can bind to lipoproteins and membranes respectively, or be degraded by phase I enzymes (Birringer M, Pfluger P, Kluth D, Landes N and Brigelius-Flohe R (2002) Identities and differences in the metabolism of tocotrienols and tocopherols in HepG2 cells. J Nutr 132:3113-3118; Neuzil J, Massa H (2005) Hepatic processing determines dual activity of vitamin E succinate. Biochem Biophys Res Commun 327:1024-1027).
Many tocopherol derivatives with a modified hydroxyl group have been tested for their pro-apoptotic activity (Table II). The most prominent derivative tested has been α-TOS (entry 1) bearing a succinylester at position C6 of the chromanol ring. Due to its low pKα (<6), α-TOS is fully deprotonated under physiological conditions, leading to a detergent-like molecule which destabilizes mitochondrial membranes and has an effect on complex II. Dicarboxylic esters of tocopherols present the best studied compounds for structure-activity relationship (SAR). Strong apoptogens included α-tocopherol succinate (1), oxalate (10), and malonate (11), the latter two inducing non-selective cytotoxicity in mice inoculated with B16-F1 melanoma cells (Kogure K, Manabe S, Suzuki I, Tokumura A and Fukuzawa K (2005) Cytotoxicity of α-tocopheryl succinate, malonate and oxalate in normal and cancer cells in vitro and their anti-cancer effects on mouse melanoma in vivo. J Nutr Sci Vitaminol 51:392-397). Even greater pro-apoptotic activity has been observed for unsaturated dicarboxylic acids like α-tocopheryl maleate (3) (Birringer M, EyTina J H, Salvatore B A and Neuzil J (2003) Vitamin E analogues as inducers of apoptosis: Structure-function relationship. Br J Cancer 88:1948-1955) and α-tocopheryl fumarate. Increasing the chain length of the dicarboxylic acid led to decreased activity as shown for glutaric acid (5), methylated glutaric acids (6, 7, 8) (Birringer et al, 2003) with the pimelic acid (24) (Kogure K, Hama S, Kisaki M, Takemasa H, Tokumura A, Suzuki I and Fukuzawa K (2004) Structural characteristic of terminal dicarboxylic moiety required for apoptogenic activity of α-tocopheryl esters. Biochim Biophys Acta 1672: 93-99) exhibiting no activity at all.
It has been established that the whole α-TOS molecule is necessary for its full apoptosis inducing activity (Birringer M, EyTina J H, Salvatore B A and Neuzil J (2003) Vitamin E analogues as inducers of apoptosis: Structure-function relationship. Br J Cancer 88:1948-1955). Esterification of the free carboxyl group leads to non-charged derivatives without pro-apoptotic activity (9, 25). Aliphatic carboxylic acid esters, such as tocopheryl acetate and propionate (19), respectively, were inactive as was the methyl ether (18). Oral administration of α-TOS is not effective since the compound is cleaved by intestinal esterases (Wu Y, Zu K, Ni J, Yeh S, Kasi D, James N S, Chemler S and Ip C (2004) Cellular and molecular effects of α-tocopheryloxybutyrate: lessons for the design of vitamin E analog for cancer prevention. Anticancer Res 24:3795-3802; Cheeseman K H, Holley A E, Kelly F J, Wasil M, Hughes L and Burton G (1995) Biokinetics in humans of RRR-α-tocopherol: the free phenol, acetate ester, and succinate ester forms of vitamin E. Free Radic Biol Med 19:591-598). To overcome the problem of ester bond cleavage, compounds (20, 21) and a side chain-truncated derivative (42) have been synthesized, replacing the ester bond with an ether bond, since the latter is resistant to hydrolysis (Wu Y, Zu K, Ni J, Yeh S, Kasi D, James N S, Chemler S and Ip C (2004) Cellular and molecular effects of α-tocopheryloxybutyrate: lessons for the design of vitamin E analog for cancer prevention. Anticancer Res 24:3795-3802; Nishikawa K, Satoh H, Hirai A, Suzuzki K, Asano R, Kumadaki I, Hagiwara K and Yano T (2003) α-Tocopheryloxybutyric acid enhances necrotic cell death in breast cancer cells treated with chemotherapy agent. Cancer Lett 201:51-56; Shun M C, Yu W, Gapor A, Parsons R, Atkinson J, Sanders B G and Kline K (2004) Pro-apoptotic mechanisms of action of a novel vitamin E analog (α-TEA) and a naturally occurring form of vitamin E (δ-tocotrienol) in MDA-MB-435 human breast cancer cells. Nutr Cancer 48:95-105; Shiau C W, Huang J W, Wang D S, Weng J R, Yang C C, Lin C H, Li C, Chen C S (2006) alpha-Tocopheryl succinate induces apoptosis in prostate cancer cells in part through inhibition of Bcl-xL/Bcl-2 function. J Biol Chem 281:11819-11825). It should be noted that the replacement of the ether bond by a methylene group is sufficient to accelerate apoptosis (22) (Sanders G., et al (2001) Preparation of tocopherols, tocotrienols, other chroman and side chain derivatives that induce cell apoptosis for therapeutic use as antiproliferative agents. 2001: PCT Int. Appl. WO 2001058889. p. 120).
When the ester bond is replaced by an amide bond, further enhancement of pro-apoptotic activity was observed (12, 13, 37, 38) (Tomic-Vatic A, EyTina J H, Chapmann J M, Mandavian E, Neuzil J and Salvatore B A (2005) Vitamin E amides, a new class of vitamin E analogues with enhanced pro-apoptotic activity. Int J Cancer 117:118-193). Again the unsaturated amides (13, 38) were superior to the saturated amides. The rationale for introducing an amide bond in place of the ester was based on the well-established fact that anilinic amides are much less prone to hydrolysis than the corresponding phenolic esters. Enhancing the stability of these tocopheryl ester derivatives would protect these molecules in vivo, allowing them to stay intact longer, thereby increasing their bioavailability. The isosteric replacement of the esters by amides makes that linkage less prone to enzymatic hydrolysis as well. Several nonspecific esterases exist in the intestinal mucosal cells and in the blood. In contrast, peptidases exhibit a much narrower specificity. For example, pro-drugs with an amino acid in an amide linkage are more stable in the intestine and blood than their corresponding ester analogues (Sugawara M, Huang W, Fei Y-J, Leibach F H, Ganaphthy V and Ganaphthy M E (2000) Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2. J Pharm Sci 89:781-789).
The last group of compounds consisted of a series of lysine α-tocopheryl esters with a positively charged N-terminus (15-17). The hydrophilic ammonium functionality exerted similar pro-apoptotic effects to its carboxylate counterpart, suggesting a general motif is required for activity that consists of a lipophilic side chain and a hydrophilic head group. However, succinyl esters of long chain aliphatic alcohols (e.g., phytol and oleol) did not show any activity (Birringer M, EyTina J H, Salvatore B A and Neuzil J (2003) Vitamin E analogues as inducers of apoptosis: Structure-function relationship. Br J Cancer 88:1948-1955).
A general SAR can be drawn from the data shown in Table II:    1. To gain profound pro-oxidant and pro-apoptotic activity, modifications of the Functional Domain I required a hydrophilic head group consisting of a dissociated acid or a charged ammonium group.    2. The chain length and the degree of unsaturation of the Functional Domain determined the apoptogenic activity. Conformational restrictions appeared to potentiate the activity.    3. The chemical linkage of the Functional Domain is not limited to esters, and other functionalities prevented enzymatic degradation of the derivatives.
The substitution pattern of the chromanol ring is often not merely related to the anti-oxidant properties of the tocopherols (Azzi A, Ricciarelli R and Zingg J M (2002) Non-antioxidant molecular functions of α-tocopherol (vitamin E). FEBS Lett 519:8-10). Different biochemical observations emphasize the role of α-tocopherol in signaling and metabolic processes. Thus, α-tocopherol is selectively recognized in the liver by α-tocopherol transfer protein (α-TTP), a 32 kDa protein with a high affinity for α-tocopherol relative to the other tocopherols and tocotrienols. The relative affinities for α-TTP decrease with the loss of methylation of the chromanol ring α-tocopherol 100%, β-tocopherol 38%, γ-tocopherol 9% and δ-tocopherol 2%) (Hosomi A, Arita M, Sato Y, Kiyose C, Ueda T, Igarashi 0, Arai H and Inoue K (1997) Affinity for α-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS Lett 409:105-108). The recently discovered tocopherol associated proteins (TAPS) show similar preferences in tocopherol binding (Yamauchi J, Iwamoto T, Kida S, Masushige S, Yamada K and Esashi T (2001) Tocopherol-associated protein is a ligand-dependent transcriptional activator. Biochem Biophys Res Commun 285:295-299). In endothelial cells, thrombin-induced PKC activation and endothelin secretion are inhibited by α-tocopherol but not by β-tocopherol (Martin-Nizard F, Boullier A, Fruchart, J C and Duriez P (1998) α-Tocopherol but not β-tocopherol inhibits thrombin-induced PKC activation and endothelin secretion in endothelial cells. J Cardiovasc Risk 5:339-345). At the transcriptional level α-tocopherol causes up-regulation of α-tropomyosin expression (Aratri E, Spycher S E, Breyer I and Azzi A (1999) Modulation of α-tropomyosin expression by α-tocopherol in rat vascular smooth muscle cells. FEBS Lett 447:91-94) and down regulation of LDL scavenger receptors SR-A and CD36, whereas β-tocopherol is ineffective (Ricciarelli R, Zingg J M and Azzi A (2000) Vitamin E reduces the uptake of oxidized LDL by inhibiting CD36 scavenger receptor expression in cultured aortic smooth muscle cells. Circulation 102:82-87; Devaraj S, Hugou I and Jialal I (2001) α-Tocopherol decreases CD36 expression in human monocyte-derived macrophages. J Lipid Res 42:521-527). In addition, the substitution pattern is likely responsible for the rate of side chain degradation because in cell culture, γ- and δ-tocopherol are degraded much faster than α- or β-tocopherol (Birringer M, Drogan D and Brigelius-Flohe R (2001) Tocopherols are metabolized in HepG2 cells by side chain ω-oxidation and consecutive β-oxidation. Free Radic Biol Med 31:226-232). Succinylation of the four tocopherol isomers produces the compounds 1, 32, 33 and 35. It is not surprising that of these, α-TOS (1) possesses the highest apoptogenic activity tested, followed by β-TOS (32), γ-TOS (33) and δ-TOS (35) as the least effective (Birringer M, Drogan D and Brigelius-Flohe R (2001) Tocopherols are metabolized in HepG2 cells by side chain ω-oxidation and consecutive β-oxidation: Free Radic Biol Med 31:226-232). In general, the more highly methylated members of the tocopherol family are the most potent, but this trend is reversed for the tocotrienols (see below).
Succinylation of Trolox, a water soluble vitamin E derivative with a shortened side chain, resulted in the complete loss of pro-apoptotic activity. SAR experiments of various tocopherol succinates bearing truncated phytol side chains (Table III, 43, 44, 45) revealed the highest level of apoptogenic activity in prostate cancer cells was obtained with derivatives where the side chain length was two isoprenyl units (43, 44). Computer assisted molecular modeling and co-immunoprecipitation experiments showed that the binding of Bak BH3 peptide to Bcl-xL and Bcl-2 was inhibited by the tocopherol analogues (Shiau C W, Huang J W, Wang D S, Weng J R, Yang C C, Lin C H, Li C, Chen C S (2006) alpha-Tocopheryl succinate induces apoptosis in prostate cancer cells in part through inhibition of Bcl-xL/Bcl-2 function. J Biol Chem 281:11819-11825). Central requirements for anti-neoplastic activity were succinylation of the chromanol ring and a minimum chain length of one isoprenyl unit (42, 46). A series of tocopheryl lysine esters with ether/ester linked Domain III side chains also showed a negative correlation between chain length and IC50 (47-50) (Arya P, Alibhai N, Qin H, Burton G W, Batist G, You S X and Alaoui-Jamali M A (1998) Design and synthesis of analogues of vitamin E: antiproliferative activity against human breast adenocarcinoma cells. Bioorg Med Chem Lett 8:2433-2438).
Tocotrienols are efficient anti-cancer agents and their pro-apoptotic property may be related to the inactivation of the Ras family of proteins. Tocotrienols exhibit their pro-apoptotic activity without modifications of the Functional Domain. The hierarchy in the Signaling Domain is also reversed, making δ-tocotrienol (59) the most potent agent in the murine B16-F10 melanoma cell model, followed by γ-(56) and α-tocotrienol (53) (Table IV; He L, Mo H, Hadisusilo S, Qureshi A A and Elson C E (1997) Isoprenoids suppress the growth of murine B16 melanomas in vitro and in vivo. J Nutr 127:668-674). Interestingly, desmethyl tocotrienol (60), lacking all aromatic methyl groups, shows even higher activity with an IC50 of 0.9 μM. This compound has been isolated from rice bran (Qureshi A A, Mo H, Packer L and Peterson D M (2000) Isolation and identification of novel tocotrienols from rice bran with hypocholesterolemic, anti-oxidant, and antitumor properties. J Agric Food Chem 48:3130-3140). A direct inhibitory action of tocotrienols has been proposed because the membrane anchoring cysteine residue of Ras proteins is modified by a common structural element, a farnesyl chain. Thus, Ras farnesylation and RhoA prenylation was inhibited by tocotrienols in A549 cells, a human lung adenocarcinoma cell line containing an activating ras mutation (Yano Y, Satoh H, Fukumoto K, Kumadaki I, Ichikawa T, Yamada K, Hagiwara K and Yano T (2005) Induction of cytotoxicity in human lung adenocarcinoma cells by 6-O-carboxy-propyl-α-tocotrienol, a redox-silent derivative of α-tocotrienol. Int J Cancer 115:839-846). To expand the short in vivo half life of tocotrienols, functional domains have been introduced. These modifications also enhanced the anti-proliferative activity of the molecules (54, 57, 58). Truncation of the side chain also improved activity, similar to that found for compound 55.
A number of compounds where modifications have been made to the Functional Domain exhibit anti-proliferative activity and provide additional specialized properties. For example, α-Tocopheryl polyethylene glycol succinate (23) has been used as a vehicle for drug delivery systems. This compound was shown to possess anti-cancer activity against human lung carcinoma cells implanted in nude mice. The apoptosis inducing efficacy of the compound was not due to its increased uptake into cells, but rather due to an increased ability to generate reactive oxygen species (Youk H J, Lee E, Choi M K, Lee Y J, Chung J H, Kim S H, Lee C H and Lim S J (2005) Enhanced anticancer efficacy of α-tocopheryl succinate by conjugation with polyethylene glycol. J Control Release 107:43-52). α-Tocopheryl phosphate (30) is believed to result from metabolism occurring during tocopherol-associated signaling (Negis Y, Zingg J M, Ogru E, Gianello R, Libinaki R and Azzi A (2005) On the existence of cellular tocopheryl phosphate, its synthesis, degradation and cellular roles: a hypothesis. IUBMB Life 57:23-25). Mixtures of 30 and di-α-tocopheryl phosphate (31) inhibited proliferation in rat aortic smooth muscle cells and in human THP-1 monocytic leukaemia cells (Munteanu A, Zingg J M, Ogru E, Libinaki R, Gianello R, West S, Negis Y and Azzi (2004) Modulation of cell proliferation and gene expression by α-tocopheryl phosphates: relevance to atherosclerosis and inflammation. Biochem Biophys Res Commun 318:311-316). The authors proposed that tocopheryl succinate and tocopheryl maleate may act in cancer cells by mimicking and substituting for tocopheryl phosphate and thereby cause the permanent activation of cellular signals.
Two experimental α-tocopheryl esters of all-trans retinoic acid (28) and 9-cis retinoic acid (29), respectively, have been used to reduce proliferation of acute promyelocytic leukaemia cells (Makishima M, Umesono K, Shudo K, Naoe T, Kishi K and Honma Y (1998) Induction of differentiation in acute promyelocytic leukemia cells by 9-cis retinoic acid α-tocopherol ester (9-cis tretinoin tocoferil). Blood 91:4715-4726). Trans-activation experiments with retinoid receptor responsive reporter constructs revealed that both of these compounds acted as agonists for retinoic acid receptors (RARs). γ-Carboxyethyl hydroxychroman (52), a degradation product of γ-tocopherol often found secreted in the urine, is able to reduce cell proliferation of PC-3 prostate cancer cells by inhibiting cyclin D1 expression (Galli F, Stabile A M, Betti M, Conte C, Pistilli A, Rende M, Floridi A and Azzi A (2004) The effect of α- and γ-tocopherol and their carboxyethyl hydroxychroman metabolites on prostate cancer cell proliferation. Arch Biochem Biophys 423:97-102).
A commonly observable difference in cancer cell compared to normal cell mitochondria is the greater mitochondrial inner trans-membrane potential (ΔΨm,i) in cancer cells. For example, as a result of the metabolic changes occurring inside cancer cells and their mitochondria, the ΔΨm,i is increased to greater negative values (−150 to −170 mV, negative inside the matrix) in carcinoma cells (Summerhayes, I. C., Lampidis, T. J., Bernal, S. D., Nadakavukaren, J. J., Nadakavukaren, K. K., Shepherd, E. L. and Chen, L. B. (1982) Unusual retention of rhodamine 123 by mitochondria in muscle and carcinoma cells. Proc Natl Acad Sci USA 79:5292-5296; Lampidis, T. J., Bernal, S. D., Summerhayes, I. C. and Chen, L. B. 1983. Selective toxicity of rhodamine 123 in carcinoma cells in vitro. Cancer Res. 43:716-720; Chen, L. B. 1988. Mitochondrial membrane potential in living cells. Annu Rev Cell Biol. 4:155-181; Modica-Napolitano, J. S. and Aprille, J. R. 1987. Basis for the selective cytotoxicity of rhodamine 123. Cancer Res. 47:4361-4365; Modica-Napolitano, J. S. and Aprille, J. R. 2001. Delocalized lipophilic cations selectively target the mitochondria of carcinoma cells. Adv Drug Deliv Rev. 49:63-70), with ˜60 mV difference across the MIM. Many proposals have been made to explain the reasons for this difference in membrane potential. At the molecular level, these include differences in mitochondrial respiratory enzyme complexes, electron carriers, ATPase, ANT and/or changes in membrane lipid metabolism. Other proposals for the increased mitochondrial membrane potential in cancer cells include altered electron transfer activity, proton translocation and utilization, or conductance. For example, mitochondria isolated from hepatocellular carcinomas display reduced uncoupler-stimulated ATP hydrolysis, decreased rates of respiration-linked ATP synthesis and reduced phosphorylating capacity compared with normal liver cells [Pedersen, P. L. and Morris, H. P. 1974. Uncoupler stimulated adenosine triphosphatase activity. Deficiency in intact mitochondria from Morris hepatomas and ascites tumor cells. J Biol. Chem. 249:3327-3334; Capuano, F., Varone, D., D'Eri, N., Russo, E., Tommasi, S., Montemurro, S., Prete, F. and Papa, S. 1996. Oxidative phosphorylation and F0F1 ATP synthase activity of human hepatocellular carcinoma. Biochem Mol Biol Int. 38:1013-1022; Capuano, F., Guerrieri, F. and Papa, S. 1997. Oxidative phosphorylation enzymes in normal and neoplastic cell growth. J Bioenerg Biomembr. 29:379-384; Cuezva, J. M., Ostronoff, L. K., Ricart, J., Lopez de Heredia, M., Di Ligero, C. M. and Izquierdo, J. M. 1997. Mitochondrial biogenesis in the liver during development and oncogenesis. J Bioenerg Biomembr. 29:365-377].
Marked changes in enzyme function, particularly in the ATPase, have been shown to occur in cancer cell mitochondria. Thus, preparations of ATPase isolated from carcinomas show reduced maximal velocity, decreased immunodetectable levels of the β subunit of the F1 component of mitochondrial ATPase and/or overexpression of the ATPase inhibitor protein (IF1) Pedersen, P. L. and Morris, H. P. 1974. Uncoupler stimulated adenosine triphosphatase activity. Deficiency in intact mitochondria from Morris hepatomas and ascites tumor cells. J. Biol. Chem. 249:3327-3334; Capuano, F., Varone, D., D'Eri, N., Russo, E., Tommasi, S., Montemurro, S., Prete, F. and Papa, S. 1996. Oxidative phosphorylation and F0F1 ATP synthase activity of human hepatocellular carcinoma. Biochem Mol Biol Int. 38:1013-1022; Capuano, F., Guerrieri, F. and Papa, S. 1997. Oxidative phosphorylation enzymes in normal and neoplastic cell growth. J Bioenerg Biomembr. 29:379-384; Cuezva, J. M., Ostronoff, L. K., Ricart, J., Lopez de Heredia, M., Di Ligero, C. M. and Izquierdo, J. M. 1997. Mitochondrial biogenesis in the liver during development and oncogenesis. J Bioenerg Biomembr, 29:365-377; reviewed in Modica-Napolitano, J. S. and Singh, K. 2002. Mitochondria as targets for detection and treatment of cancer. Expert Rev Mol. Med. 2002:1-19). A reduced ability to use the proton gradient to make ATP, with a resulting build up in the protons within the MIM would account for the greater ΔΨm,i existing in tumour mitochondria. Another possibility that may account for greater ΔΨm,i in cancer cells is that acetoin undergoes an ATP dependent reaction, almost doubling the reaction rate to produce citrate in tumour cells (Baggetto, L. G. and Lehninger, A. L. 1987. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes. Biochem Biophys Res Commun. 145:153-159; Baggetto, L. G. and Testa-Parussini, R. 1990. Role of acetoin on the regulation of intermediate metabolism of Ehrlich ascites tumor mitochondria: its contribution to membrane cholesterol enrichment modifying passive proton permeability. Arch Biochem Biophys. 283:241-248), which is then exported by the tricarboxylate or citrate carrier (CIC) to the cytosol where it is cleaved to oxaloacetate and acetyl-coA. The net effect is the provision of a high level of cytoplasmic acetyl-coA precursor for sterol biosynthesis, particularly helping to promote the already elevated cancer cell production of cholesterol (Baggetto, L. G. and Testa-Parussini, R. 1990. Role of acetoin on the regulation of intermediate metabolism of Ehrlich ascites tumor mitochondria: its contribution to membrane cholesterol enrichment modifying passive proton permeability. Arch Biochem Biophys. 283:241-248). The resulting build-up of cholesterol in the inner MIM reduces several fold their passive proton permeability, helping to establish the greater transmembrane potential in cancer cells (Baggetto, L. G. and Testa-Parussini, R. 1990. Role of acetoin on the regulation of intermediate metabolism of Ehrlich ascites tumor mitochondria: its contribution to membrane cholesterol enrichment modifying passive proton permeability. Arch Biochem Biophys. 283:241-248; Baggetto, L. G. 1992. Deviant energetic metabolism of glycolytic cancer cells. Biochimie. 74:959-974).
The enhanced glycolytic activity due to very high energetic demand increases cytoplasmic levels of lactic acid production in cancer cells. To maintain the neutral pH of the cytosol these cells activate plasma membrane proton pumps causing extracellular acidification. Typically, the pH of the tumour interstitium is 6.2-6.5, while the pH of normal tissue interstitium is neutral (Gerweck, L. E. 2000. The pH difference between tumor and normal tissue offers a tumor specific target for the treatment of cancer. Drug Resist Updat. 3:49-50; Gerweck, L. E., Vijayappa, S. and Kozin, S. 2006. Tumor pH controls the in vivo efficacy of weak acid and base chemotherapeutics. Mol Cancer Ther. 5:1275-1279). The major type of proton pumps used by cancer cells to maintain their neutral cytosolic pH is the class V ATPase (Sennoune, S. R., Luo, D. and Martinez-Zaguilan, R. 2004. Plasmalemmal vacuolar-type H+-ATPase in cancer biology. Cell Biochem Biophys. 40:185-206). This ATPase has relatively low activity in non-malignant cells, while its activity is increased in cancer cells (Izumi, H., Torigoe, T., Ishiguchi, H., Uramoto, H., Yoshida, Y, Tanabe, M., Ise, T., Murakami, T., Yoshida, T., Nomoto, M. and Kohno, K. 2003. Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy. Cancer Treat Rev. 29:541-549; Bowman, E. J. and Bowman, B. J. 2005. V-ATPases as drug targets. J Bioenerg Biomembr. 37:431-435). These observations led to development of a novel anti-cancer strategy by inhibiting the proton pumping activity of the ATPase, causing acidification of the cancer cell cytosol that, in turn, results in the demise of the cell. For example, chondropsin compounds (Bowman, E. J., Gustafson, K. R., Bowman, B. J. and Boyd, M. R. 2003. Identification of a new chondropsin class of antitumor compound that selectively inhibits V-ATPases. J Biol. Chem. 278: 44147-44152) and siRNA targeting the ATPase subunit ATP6L (Lu, X., Qin, W., Li, J., Tan, N., Pan; D., Zhang, H., Xie, L., Yao, G., Shu, H., Yao, M., Wan, D., Gu, J. and Yang, S. (2005) The growth and metastasis of human hepatocellular carcinoma xenografts are inhibited by small interfering RNA targeting to the subunit ATP6L of proton pump. Cancer Res. 65:6843-6849) have been successfully used to kill cancer cells. Other important regulators of cytosolic pH are the Na+/H+ antiporter (Slepkov, E. R., Rainey, J. K., Sykes, B. D. and Fliegel, L. 2007. Structural and functional analysis of the Na+/H+ exchanger. Biochem J. 401:623-633), the H+/lactate symporter (Cardone, R. A., Casavola, V. and Reshkin, S. J. 2005. The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer. 5:786-795), and the Na+-dependent Cl−/HCO-exchanger (Lee, A. H. and Tannock, I. F. 1998. Heterogeneity of intracellular pH and of mechanisms that regulate intracellular pH in populations of cultured cells. Cancer Res. 58:1901-1908). Similarly as for V-class ATPase, these transporters have been proposed as targets for anticancer drugs (Izumi, H., Torigoe, T., Ishiguchi, H., Uramoto, H., Yoshida, Y., Tanabe, M., Ise, T., Murakami, T., Yoshida, T., Nomoto, M. and Kohno, K. 2003. Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy. Cancer Treat Rev. 29:541-549 Cardone, R. A., Casavola, V. and Reshkin, S J 2005. The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer. 5:786-795; Harguindey, S., Orive, G., Luis Pedraz, J., Paradiso, A. and Reshkin, S. J. 2005. The role of pH dynamics and the Na+/H+ antiporter in the etiopathogenesis and treatment of cancer. Two faces of the same coin—one single nature. Biochim Biophys Acta. 1756:1-24).
The difference in pH gradient across the plasma membrane of cancer cells has been used to design a class of drugs that can be classified as weak acids, with pKa values of <6, and which are, typically, deprotonated at neutral pH but accept a proton at the pH of the tumour interstitium (Gerweck, L. E., Vijayappa, S. and Kozin, S. 2006. Tumor pH controls the in vivo efficacy of weak acid and base chemotherapeutics. Mol Cancer Ther. 5:1275-1279). A prototypic example of such a drug is the weak acid chlorambucil (Skarsgard, L. D., Chaplin, D J., Wilson, D. J., Skwarchuk, M. W., Vinczan, A. and Kristl, J. 1992. The effect of hypoxia and low pH on the cytotoxicity of chlorambucil. Int J Radiat Oncol Biol Phys. 22:737-741). It was reported that the relatively selective uptake and anti-cancer efficacy of chlorambucil could be enhanced by injection of glucose into mice with experimental tumours, thereby further promoting glycolytic activity of tumour cells, lowering the tumour interstitium pH while not affecting the pH of the tumour cell cytosol (Kozin, S. V., Shkarin, P. and Gerweck, L. E. (2001) The cell transmembrane pH gradient in tumors enhances cytotoxicity of specific weak acid chemotherapeutics. Cancer Res. 61:4740-4743).
The present inventors have observed similar effects for the mitocan α-tocopheryl succinate (α TOS), a compound with pKa of 5.6 (Neuzil, J., Zhao, M., Ostermann, G., Sticha, M., Gellert, N., Weber, C., Eaton, J. W. and Brunk, U. T. 2002 α-Tocopheryl succinate, an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability. Biochem J. 362:709-715). The vitamin E analogue is a weak acid, of which ˜98% is deprotonated at neutral pH with 10-15-fold higher percentage in the protonated form at the acidic pH of 6.2-6.4 of the tumour interstitium. Since there are no known transporters for compounds like α-TOS, presumably it crosses the plasma membrane to freely diffuse inside the cells and discharge its apoptogenic activity. Accordingly, the present inventors have found that when the pH of the tissue culture medium was more acidic (pH ˜6.2), it resulted in ˜3-times greater apoptogenic efficacy of α-TOS against T lymphoma cells compared to media at pH of 7.2. The likely reason is the faster uptake of the compound at lower pH, since it was found that about twice as much α-TOS enters at pH 6.2 than when the pH of the medium was 7.4 (Neuzil, J., Zhao, M., Ostermann, G., Sticha, M., Gellert, N., Weber, C., Eaton, J. W. and Brunk, U. T. 2002 α-Tocopheryl succinate, an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability. Biochem J. 362: 709-715). Thus, the pH differential across tumour plasma membranes may be an important paradigm for targeted delivery whereby certain anticancer agents exert selectivity for malignant tissues.
The sixth class of mitocans listed in Table I includes molecules that are delocalized lipophilic cations which accumulate at much greater concentrations in the mitochondrial matrix than in the cytoplasm of cells (Smith, R. A., Porteous, C. M., Gane, A. M. and Murphy, M. P. 2003. Delivery of bioactive molecules to mitochondria in vivo. Proc Natl Acad Sci USA 100:5407-5412). These agents are selectively accumulated in the mitochondrial matrix of cancer cells because of their greater transmembrane potentials across the plasma membrane as well as their more polarized mitochondria with a much greater ΔΨm,i than that in non-malignant cells (Davis, S., Weiss, M. J., Wong, J. R., Lampidis, T. J. and Chen, L. B. 1985. Mitochondrial and plasma membrane potentials cause unusual accumulation and retention of rhodamine 123 by human breast adenocarcinoma-derived MCF-7 cells. J Biol. Chem. 260:13844-13850; Lampidis, T. J., Hasin, Y, Weiss, M. J. and Chen, L. B. 1985. Selective killing of carcinoma cells “in vitro” by lipophilic-cationic compounds: a cellular basis. Biomed Pharmacother. 39:220-226; Ralph, S. J., Low, P., Dong, L., Lawen, A. and Neuzil, J. 2006. Mitocans: mitochondrial targeted anticancer drugs as improved therapies and related patent documents. Recent Patents Anti-cancer Drug Discovery 1:327-346).
The target for the lipophilic cation-based mitocans may be one of the inhibitory binding sites on ATPase (Gledhill, J. R. and Walker, J. E. 2005. Inhibition sites in F1-ATPase from bovine heart mitochondria. Biochem J. 386:591-598). One of the earliest members of this class of compounds to be identified for its anti-cancer activity was rhodamine-123 (Bernal, S. D., Lampidis, T. J., Summerhayes, I. C. and Chen, L. B. 1982. Rhodamine-123 selectively reduces clonal growth of carcinoma cells in vitro. Science 218:1117-1119; Bernal, S. D., Lampidis, T. J., Mclsaac, R. M. and Chen, L. B. 1983. Anti-carcinoma activity in vivo of rhodamine 123, a mitochondrial-specific dye. Science 222:169-172). It recently entered phase I clinical trials for prostate cancer and revealed minimal side effects and safe administration at monthly intervals without detectable drug accumulation in the serum of patients (Jones, L. W., Narayan, K. S., Shapiro, C. E. and Sweatman, T. W. 2005. Rhodamine-123: therapy for hormone refractory prostate cancer, a phase I clinical trial. J Chemother. 17:435-440). It is likely that the related compound, Rose Bengal, works in a similar fashion to rhodamine 123, and Rose Bengal is likewise currently in clinical trials as a therapy for metastatic melanoma and recurrent breast cancer, causing complete remissions in some patients (Provectus PV-10-MM-01, www.ClinicalTrials.gov).
The drug F16 is a mechanistically more characterized example of this mitocan class and was shown to increase ROS production, depolarize mitochondria as a weak protonophore and collapse ΔΨm,i leading to mitochondrial permeability transition and selective apoptosis of cancer cells when applied in the micromolar range (Fantin, V. R., Berardi, M. J., Scorrano, L., Korsmeyer, S. J. and Leder, P. (2002) A novel mitochondriotoxic small molecule that selectively inhibits tumor cell growth. Cancer Cell 2:29-42). F16 was also reported in the study to inhibit the growth of mammary tumours in mice. MKT-077, a rhodocyanine dye analogue is another example of this type of mitocan that entered phase I clinical trials, although these were terminated due to renal toxicity (Britten, C. D., Rowinsky, E. K., Baker, S. D., Weiss, G. R., Smith, L., Stephenson, J., Rothenberg, M., Smetzer, L., Cramer, J., Collins, W., Von Hoff, D. D. and Eckhardt, S. G. 2000. A phase I and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog MKT 077. Clin Cancer Res. 6:42-49).
This raises the issue of toxicity with many of the class VI mitocans. A cautionary example of the potential for toxicity that must be carefully evaluated is the production of Parkinson's-like effects by the drug MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) caused by the selective destruction of the nigrostriatal dopaminergic neurons. This toxicity was due to the selective uptake by the dopamine transporters on these cells as well as metabolites formed by the action of the enzyme, monoamine oxidase B that is highly expressed in the dopaminergic neurons. As a result of these two unique properties of dopaminergic neurons, the mitochondriotoxic drug MPP+ (1-methyl-4-phenylpyridinium) is produced which acts as an inhibitor of mitochondrial respiration by blocking the NADH-ubiquinone oxidoreductase site of complex I. It is likely that MPP+ is also a protonophore (Davey, G. P., Tipton, K. F. and Murphy, M. P. 1992. Uptake and accumulation of 1-methyl-4-phenylpyridinium by rat liver mitochondria measured using an ion-selective electrode. Biochem J. 288:439-443; Albores, R., Neafsey, E J., Drucker, G., Fields, J. Z. and Collins, M. A. 1990. Mitochondrial respiratory inhibition by N-methylatedβ-carboline derivatives structurally resembling N-methyl-4-phenylpyridine. Proc Natl Acad Sci USA 87:9368-9372) that collapses the ΔΨm,i leading to cell destruction. This, together with the non-selective cell toxicity associated with another lipophilic cation and known mitochondrial poison, dequalinium chloride (Gamboa-Vujicic, G., Emma, D. A., Liao, S. Y, Fuchtner, C. and Manetta, A. 1993. Toxicity of the mitochondrial poison dequalinium chloride in a murine model system. J Pharm Sci. 82:231-235), raises the importance of identifying class VI mitocans that are cancer cell-specific in terms of their uptake and cellular toxicity, as recently described in a predictive model based on their structures by Trapp, S. and Horobin, R. W. (2005. A predictive model for the selective accumulation of chemicals in tumor cells. Eur Biophys J. 34:959-966).
The amphipathic and positively charged α-helical pro-apoptotic peptide (KLAKLAK)2 has also been included in this class of mitocans as a delocalized lipophilic cation. However, the peptide must first be coupled to a targeted delivery system for surface receptor binding and uptake into cancer cells, before it is able to function as a mitocan (Ellerby, H. M., Arap, W., Ellerby, L. M.; Kain, R., Andrusiak, R., Rio, G. D., Krajewski, S., Lombardo, C. R., Rao, R., Ruoslahti, E., Bredesen, D. E. and Pasqualini, R. 1999. Anti-cancer activity of targeted pro-apoptotic peptides. Nat. Med. 5:1032-1038; Fantin et al. 2005). As with the other members of this class of mitocans, the peptide has been shown to dissipate ΔΨm,i leading to apoptosis, and it efficiently reduced tumour burdens in animal models (Fantin, V. R., Berardi, M. J., Babbe, H., Michelman, M. V., Manning, C. M. and Leder, P. 2005. A bifunctional targeted peptide that blocks HER-2 tyrosine kinase and disables mitochondrial function in HER-2-positive carcinoma cells. Cancer Res. 65:6891-6900):